Roberto Manetti

The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin

The VacA cytotoxin, produced by toxigenic strains of Helicobacter pylori, induces the formation of large vacuoles highly enriched in the small GTPase rab7. To probe the role of rab7 in vacuolization, HeLa cells were transfected with a series of rab mutants and exposed to VacA. Dominant‐negative mutants of rab7 effectively prevented vacuolization, whereas homologous rab5 and rab9 mutants were only partially inhibitory or ineffective, respectively. Expression of wild‐type or GTPase‐deficient rab mutants synergized with VacA in inducing vacuolization. In vitro fusion of late endosomes was enhanced by active rab7 and inhibited by inactive rab7, consistent with vacuole formation by merging of late endosomes in a process that requires functional rab7. Taken together, the effects of overexpressed rab proteins described here indicate that continuous membrane flow along the endocytic pathway is necessary for vacuole growth.

LOW PH ACTIVATES THE VACUOLATING TOXIN OF HELICOBACTER PYLORI, WHICH BECOMES ACID AND PEPSIN RESISTANT

The protein toxin VacA, produced by cytotoxic strains of Helicobacter pylori, causes a vacuolar degeneration of cells, which eventually die. VacA is strongly activated by a short exposure to acidic solutions in the pH 1.5-5.5 range, followed by neutralization. Activated VacA has different CD and fluorescence spectra and a limited proteolysis fragmentation pattern from VacA kept at neutral pH. Moreover, activated VacA is resistant to pH 1.5 and to pepsin. The relevance of these findings to pathogenesis of H. pylori-induced gastrointestinal ulcers is discussed.

Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.

PROBING THE STRUCTURE-ACTIVITY RELATIONSHIP OF ESCHERICHIA COLI LT-A BY SITE-DIRECTED MUTAGENESIS

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat‐labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site‐directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non‐toxic new molecules containing mutations of single amino acids such as Val‐53 → Glu or Asp, Ser‐63 → Lys, Val‐97 → Lys, Tyr‐104 → Lys or Asp, and Ser‐14 → Lys or Glu. This group also included mutations in amino acids such as Arg‐7, Glu‐110 and Glu‐112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu‐41 → Phe, Ala‐45 → Tyr or Glu, Val‐53 → Tyr, Val‐60 → Gly, Ser‐68 → Pro, His‐70 → Pro, Val‐97 → Tyr and Ser‐114 → Tyr. The third group contained those molecules that maintained a wild‐type level of toxicity in spite of the mutations introduced: Arg‐54 → Lys or Ala, Tyr‐59 → Met, Ser‐68 → Lys, Ala‐72 → Arg, His or Asp and Arg‐192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non‐toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.

A novel chromatin-forming histone H1 homologue is encoded by a dispensable and growth-regulated gene in Bordetella pertussis

We report the Identification of a protein homologous to a histone H1 in Bordetella pertussis. The B. pertussis histone homologue, BpH1, varies in size in different strains from 182 to 206 amino acids. The variability of the size of the protein is due to gene variability by insertion or deletion of DNA modules. Insertion of a kanamycin cassette into the bpH1 gene generates a BpH1 null mutant with phenotypic properties and growth rate similar to those of the wild‐type strain, showing that this gene is dispensable. In vitro, the BpH1 protein prevents chromosomal DNA degradation from DNase I and constrains supercoiled DNA. Transcription of the bpH1 gene is activated during exponential growth of the bacteria, whereas It is repressed during the stationary phase of growth, It is proposed that BpH1 plays a role in chromatin formation and condensation during DNA replication and that repression of transcription depends upon a reduced rate of DNA replication.

Development and clinical testing of an acellular pertussis vaccine containing genetically detoxified pertussis toxin

In 1924 Ramon described the inactivation of diphtheria toxin by formaldehyde treatment. This method allowed the introduction of mass vaccination against diphtheria and tetanus and opened the way to the inactivation of viruses by chemical treatment. In this review we describe the use of genetic manipulations for the inactivation of pertussis toxin. The toxin inactivated by this new method is an antigen superior to those obtained by chemical treatment and has been used to develop a new vaccine against whooping cough.

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Abstract Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica.